Nested polymerase chain reaction for detection of Ehrlichia equi genomic DNA in horses and ticks (Ixodes pacificus)

Jeffrey E. Barlough, John E Madigan, Elfriede DeRock, Luisa Bigornia

Research output: Contribution to journalArticle

124 Citations (Scopus)

Abstract

A nested polymerase chain reaction for detecting Ehrlichia equi in horses and ticks (Ixodes pacificus) was developed. A major second-round PCR product of 928 bp could be readily visualized in ethidium bromide-stained agarose minigels. An internal probe was used to verify the identity of the amplified product by non-radioactive (digoxigenin-based) Southern blotting; additional confirmation was provided by DNA sequence analysis. A dilution study testing the sensitivity of the PCR indicated that DNA derived from ≤ 7.6 but > 3 infected neutrophils was sufficient to generate a PCR signal. The specificity of the PCR was examined using a panel of rickettsiae, of which only E. equi and the closely-related human granulocytotropic ehrlichia produced PCR bands. In an in vivo infection study, E. equi DNA was detected in blood buffy-coat cells of an experimentally-infected horse on days three through 14 post- inoculation. In a separate study, three of six adult I. pacificus that as nymphs had been fed on an experimentally infected horse were found to be PCR- positive for E. equi.

Original languageEnglish (US)
Pages (from-to)319-329
Number of pages11
JournalVeterinary Parasitology
Volume63
Issue number3-4
DOIs
StatePublished - Jun 1996

Fingerprint

Ixodes pacificus
Anaplasma phagocytophilum
Ixodes
Ticks
Horses
ticks
polymerase chain reaction
genomics
horses
Polymerase Chain Reaction
DNA
Ehrlichia
digoxigenin
Southern blotting
agarose
Blood Buffy Coat
nymphs
neutrophils
sequence analysis
Digoxigenin

Keywords

  • Diagnosis Rickettsia
  • DNA sequence analysis
  • Ehrlichia equi
  • Horse
  • Ixodes pacificus
  • Polymerase chain reaction
  • Southern hybridization test

ASJC Scopus subject areas

  • Animal Science and Zoology
  • Parasitology
  • veterinary(all)

Cite this

Nested polymerase chain reaction for detection of Ehrlichia equi genomic DNA in horses and ticks (Ixodes pacificus). / Barlough, Jeffrey E.; Madigan, John E; DeRock, Elfriede; Bigornia, Luisa.

In: Veterinary Parasitology, Vol. 63, No. 3-4, 06.1996, p. 319-329.

Research output: Contribution to journalArticle

Barlough, Jeffrey E. ; Madigan, John E ; DeRock, Elfriede ; Bigornia, Luisa. / Nested polymerase chain reaction for detection of Ehrlichia equi genomic DNA in horses and ticks (Ixodes pacificus). In: Veterinary Parasitology. 1996 ; Vol. 63, No. 3-4. pp. 319-329.
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